Diagnostic systems such as biosensors have been extensively studied as a result of human desires to overcome diseases and live longer by monitoring the diseases at an early stage. Many commercialized diagnostic systems are based on antigen-antibody immune reactions or ligand-acceptor interactions. However, certain parts of the diseases actually developed in humans may not be detected by such antigen-antibody reactions or ligand-acceptor reactions. In addition, detection systems capable of highly sensitive or quantitative detection are required when detection of micromaterials is needed or quantitation of specific materials, rather than the information on the presence thereof, is needed. The most widely used method for detecting specific biomaterials is labeling a fluorescent material and detecting the fluorescence released therefrom at a specific wavelength. However, such a method of using fluorescence has disadvantages in that 1) a process of labeling a fluorescent substance to a target material is required, and 2) the fluorescent substance exhibits blinking or is quenched in process of time or due to light exposure. Accordingly, as a result of noting that oxidation and reduction are involved in various mechanisms occurring in vivo and that superoxide may act as a main marker for diseases such as cancers, diabetes, Alzheimer's disease or Parkinson's disease, methods of detecting electrochemical signals generated from these processes or from superoxide as a product have emerged as new diagnostic methods.
Cytochrome c (cyt c) is a typical protein capable of detecting superoxide (O2−) and nitric oxide (NO), and gives signals capable of detecting diseases such as cancers, diabetes, Alzheimer's disease and Parkinson's disease. The most widely used principle in such cyt c-based devices is to monitor electron transfer of cyt c molecules when oxidation and reduction processes for specific interactions with external molecules occur. Accordingly, immobilizing cyt c molecules on an electrode through stable binding is very important in order to acquire reliable signals during interactions, that is, a detection process.
Two most reasonable methods for binding cyt c molecules to an electrode reported to date are (1) binding by coating a mixture of cyt c molecules and a polymeric binder, which is simple and cost-effective, and (2) binding through charge interaction between cyt c molecules and an electrode surface since cyt c molecules have polar functional groups such as amine (—NH2), carboxylic acid (—COOH), and amide bonds (—CONH—). However, these methods only deliver a minimal binding state (for example, physical adhesion), and may not fundamentally render a strong binding force as in covalent bonds.
In view of the above, the inventors of the present invention have carried out extensive research in order to more stably immobilize biomolecules such as protein and DNA (for example, cyt c molecules, a metal-containing heme protein or the like) capable of delivering electrical signals, which are generated in a redox process occurring in vivo, on the surface of an electrode, and as a result, identified that more desirable cyt c molecule immobilization on the surface of an electrode may be accomplished through an anchoring role by covalently bonding the biomolecules using an organic coupler comprising 2 or more carboxylic acid groups, such as pyridine dicarboxylic acid (PDA), as a linker, and completed the present invention (refer to FIG. 1a). In particular, the inventors of the present invention have demonstrated that cyt c molecules may be densely and stably immobilized on the surface of an electrode when a mesoporous titania (titanium dioxide) coating layer is formed on the electrode, since each titania pore may contain 1 or 2 cyt c molecules therein, and covalent bonds with carboxylic acid groups of a PDA coupler may be formed by an esterification reaction using a Ti—OH functional group present abundantly on the titania surface as well.